Association between amorphous calcium-phosphate ratios in circulating calciprotein particles and prognostic biomarkers in hemodialysis patients

Calciprotein particles (CPPs) are circulating colloidal mineral-protein complexes containing crystalline and/or non-crystalline (amorphous) calcium-phosphate (CaPi). Serum CPP levels correlate with vascular stiffness and calcification in patients with chronic kidney disease (CKD). In vitro studies showed that CPPs containing crystalline CaPi were more arteriosclerogenic and inflammogenic than CPPs without containing crystalline CaPi. Thus, we hypothesized that not only the quantity but also the quality of CPPs (the phase of CaPi) might affect clinical outcomes. To test this hypothesis, we quantified amorphous CaPi ratio defined as the ratio of the amorphous CaPi amount to the total CaPi amount in serum CPPs from 183 hemodialysis patients and explored its possible correlation with serum parameters associated with prognosis of hemodialysis patients. Multivariate analysis revealed that the amorphous CaPi ratio correlated positively with hemoglobin and negatively with fibroblast growth factor-21 (FGF21), which remained significant after adjusting for the total CaPi amount. Because low hemoglobin and high FGF21 are associated with increased mortality, the present study warrants further studies to determine whether low amorphous CaPi ratio in circulating CPPs may be associated with poor prognosis in hemodialysis patients.


Discussion
Serum FGF21 levels are consistently increased during CKD progression since early stages 10,11 . FGF21 is a member of the fibroblast growth factor (FGF) subfamily that functions as an endocrine factor 12 . A characteristic feature of FGF21 lies in the fact that it requires βKlotho, a single-pass transmembrane protein, to bind to FGF receptor-1c (FGFR1c) 13,14 . Namely, the physiological receptor for FGF21 is not an FGF receptor but a binary complex of FGFR1c and βKlotho, which is expressed predominantly in adipocytes and neurons in the suprachiasmatic nucleus (SCN) 15 . In response to various types of stress including fasting, FGF21 is secreted from hepatocytes and acts directly on adipocytes and indirectly on the liver to induce metabolic responses to fasting 16,17 . In Table 1. Clinical and laboratory characteristics of the participants. Data are expressed as mean ± SD or number or median (interquartile range). BMI body mass index, SBP systolic blood pressure, DBP diastolic blood pressure, iPTH intact parathyroid hormone, BAP bone specific alkaline phosphatase, TSAT transferrin saturation, CRP C-reactive protein.  www.nature.com/scientificreports/ addition, FGF21 passes through the blood-brain barrier and acts directly on SCN neurons to activate the sympathetic nervous system and the hypothalamus-pituitary-adrenal axis, thereby inducing responses to stress 18 . The increase in FGF21 during CKD progression can be viewed as a survival response, because CKD mice lacking FGF21 have shorter lifespan than wild-type CKD mice 19 . Thus, high serum FGF21 levels may indicate a state of intense stress and is indeed associated with poor prognosis in hemodialysis patients 20 . Conversely, the fact that the hemodialysis patients with higher amorphous CaPi ratios had lower serum FGF21 levels ( Table 5) suggests that they may have better prognosis. This notion is also supported by the fact that the amorphous CaPi ratio was positively correlated with hemoglobin, because higher hemoglobin was reported to be associated with better prognosis in CKD patients 21 . Serum FGF23 levels were reported to correlate with mortality independently of serum phosphate levels in hemodialysis patients 22 . Although we were unable to measure FGF23 in this study due to the limitation of the sample volume, we speculate that FGF23 may correlate positively with amorphous and/or crystalline CaPi contents, because CPPs stimulated FGF23 secretion/production in cultured osteoblastic cells 23 . Indeed, we previously reported that high crystalline CaPi amounts were associated with high serum FGF23 levels in hemodialysis patients 24 . Chronic non-infectious inflammation is also known to be associated with poor prognosis in Table 3. Univariate and multivariate analysis between crystalline CaPi amounts and serum parameters in the hemodialysis patients. Significant values are in bold. BMI body mass index, PTH parathyroid hormone, BAP bone specific alkaline phosphatase, TRACP-5b tartrate-resistant acid phosphatase-5b, TSAT transferrin saturation, CRP C-reactive protein.  www.nature.com/scientificreports/ hemodialysis patients 25 . However, we were unable to detect significant correlation between CRP and any types of CPPs in multivariate analysis in this study. Serum phosphate levels were positively associated not only with amorphous CaPi amounts but also crystalline CaPi amounts ( Fig. 2A,B). Crystalline CaPi amounts reflect secondary CPPs with pathogenic activity that induces calcification in cultured vascular smooth muscle cells, whereas amorphous CaPi amounts reflect primary CPPs. There is no experimental evidence showing that primary CPPs exert the pathogenic activity like secondary CPPs or counteract the pathogenic activity of secondary CPPs. Therefore, we assume that secondary CPPs, but not primary CPPs, may contribute to poor clinical outcomes associated with hyperphosphatemia.
In general, insoluble materials such as lipids and CaPi are bound to specific serum proteins to form colloidal particles and dispersed in the blood to be transported between organs through systemic circulation. Lipids are bound to apoproteins to form colloidal particles called lipoproteins. The activity of lipoproteins depends on their composition and physical properties, as evidenced by the fact that low-density lipoprotein (LDL) is pro-atherogenic, whereas high-density lipoprotein (HDL) is anti-atherogenic 26 . Thus, not only total cholesterol levels but also lipoprotein fractions have been measured for evaluating the risk for atherosclerosis. Similarly, insoluble CaPi is bound to fetuin-A to form colloidal particles called CPPs. The activity of CPPs depends on their composition and physical properties, as evidenced by the fact that secondary CPPs, but not primary CPPs, exert cytotoxic activity 27 . Thus, we propose that measurement of both quantity (serum levels) and quality (amorphous CaPi ratios) of CPPs may be informative for evaluating prognosis and clinical outcomes in hemodialysis patients.
One of the limitations in this study is lack of a cohort of healthy individuals. Unlike in hemodialysis patients, CPP levels were barely increased after incubation at 25 °C for 24 h in healthy volunteers 8 . Therefore, we presume that measurement of amorphous CaPi amounts and amorphous CaPi ratios using the gel filtration assay may not be feasible in healthy individuals. Other limitations include a cross-sectional design and a small number of participants. Further long-term, large-scale, prospective cohort studies are needed to conclude that the amorphous CaPi ratio serves as an independent parameter that predicts prognosis in hemodialysis patients.

Methods
Patients. Total of 183 end-stage renal disease (ESRD) patients receiving hemodialysis (57.6% men, median age 71, range 41-100 years) were recruited in a single hospital (St. Hill hospital, Ube, Yamaguchi, Japan). The exclusion criteria were active malignancy and severe infectious disease. Patient's case history and comorbidities were obtained from medical records. The causes of kidney disease included diabetic nephropathy (33%), hypertensive nephrosclerosis (24%), chronic glomerular nephritis (20%), polycystic kidney disease (2%), and others (21%). Comorbidities included hypertension (82%), diabetes mellitus (39%), cardiovascular disease defined as history of cardiac and cerebrovascular (including stroke) events (40%). The study was conducted in accordance with the Declaration of Helsinki. The research protocol was approved by the Medical Ethics Committees of St. Hill hospital. All the patients provided their written informed consent.
Data collection. Blood samples were collected from patients immediately before starting a hemodialysis session. Serum FGF21 levels were measured using a sandwich enzyme-linked immunosorbent assay (ELISA) kit (Bio Vender, Mpdrice, Czech Republic) in accordance with the manufacturer's instructions. Other laboratory data were measured using certified methods at the Department of Clinical Chemistry of the hospital.